The blood complement (C) plays an important role in host defense to foreign substances. Individuals that are deficient in certain C components often Buffer recurrent and sometimes fatal infections. Activation of the C system results in the production of the anaphylatoxins, C3a and C5a. These fragments are biologically active cleavage products of the larger C proteins C3 and C5, respectively. C5a is a short (74 residues in human) glycoprotein that is generated by enzymatic cleavage of C5.
C5a is recognized as a principal mediator of local and systemic inflammatory responses because of its ability to activate and recruit neutrophils, induce spasmogenesis, increase vascular permeability and stimulate the release of secondary inflammatory mediators from a variety of cell types (e.g., leukocytes and macrophages). C5a also appears to play a role in the modulation of immune response because of its ability to induce, directly or indirectly, the synthesis and release of the cytokines interleukin-1 (IL-1), interleuken-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-.alpha. (TNF-.alpha.) from human monocytes. These inflammatory and immunomodulatory activities are believed to be expressed via a transmembrane, G-protein-mediated signal transduction mechanism when the C5a ligand interacts with its receptor(s) expressed on the surface of certain circulating and tissue cell types.
The proinflammatory activities of C5a may be classified into two broad categories. The first category of activity is generally associated with the release of histamines and other secondary mediators (e.g., vasoconstrictor and vasodilator eicosanoids). These activities of C5a affect many systems, and are associated with the phenomena of spasmogenesis and certain cell aggregatory activities (e.g., platelet aggregation). The second category of activity involves recruitment and activation of neutrophils and subsequent effects of such neutrophil accumulation and activation, such as increased vascular permeability, release of cytokines and other pro-inflammatory responses. The in vivo pharmacology of these two broad classes of C5a activities is described briefly by Drapeau et al. (1993), Biochem. Pharmacol., 45: 1289-1299. The regulation of neutrophils and other leukocytes by C5a has been reviewed by Hugli & Morgan (1984), Chapter 4 in Regulation of Leukocyte Function, R. Snyderman, ed., Plenum Publishing Corp., pp. 109-153.
Because of its proinflammatory activity, C5a has been implicated as a pathogenic factor in the expression of certain inflammatory disorders, such as rheumatoid arthritis, adult respiratory distress syndrome, gingivitis, and the tissue damage associated with atherosclerosis and myocardial infarction. Consequently, considerable research efforts have been expended in developing specific C5a antagonists for use as anti-inflammatory agents in the treatment of these diseases.
One approach to the development of a potent C5a antagonist has focused on the synthetic manipulation of peptides possessing sequence homology to the C-terminal "effector" region of C5a. These peptides have been shown to be effective agonists compared to the parent polypeptide, but at markedly reduced potencies (see, e.g., Ember et al. (1994), Amer. J. Pathol., 144: 393-403; Ember et al. (1992), J. Immunol., 148: 3165-3173; Morgan et al. (1992), J. Immunol., 148: 3937-3942). Therefore, a first step toward the development of an antagonist would be to increase the potency of these agonist peptides to a level approaching that of natural C5a, the rationale being that the increase in potency reflects a heightened affinity for the C5a receptor. Such potent peptide agonists could be used as templates from which an analog or mimetic is developed that would retain the high affinity binding characteristics, so as to compete with natural C5a for the receptor, but not transduce a biological signal when bound to the receptor.
On the other hand, C-terminal agonists of C5a have been shown to induce the synthesis and release of several immune-modulatory cytokines from human monocytes (see Goodman et al. (1982), J. Immunol., 129: 70-75; Okusawa et al. (1987), J. Immunol., 139: 2635-2639; Scholz et al. (1990), Clin. Immunol. Immunopathol., 57: 297-307; Ember et al. (1994), Amer. J. Pathol., 144: 393-403). Because of its multiple roles in the cellular and humoral immune response, considerable interest also exists in developing specific C5a agonists as immune adjuvants for treatment of immunocompromised patients.
Ideally, C5a agonists or antagonists would not only be potent, but would be selective for a specific desired biological response associated with naturally-occurring C5a. For example, an analog that could stimulate the immune-modulatory effect in monocytes at the expense of other C5a-mediated inflammatory responses would have considerable therapeutic utility as an immune adjuvant for stimulating cellular and humoral immune responses, but exhibiting no inflammatory side effects. As another example, C5a has been shown to have a direct effect on rat pulmonary artery endothelial cells, implying the presence of functional C5a receptors on these and other endothelial tissues (Friedl et al. (1989), FASEB J., 3: 2512-2518; Ward (1991), Am. J. Med., 91 (Suppl. 3C): 89S-94S). Accordingly, another therapeutic utility for a selective agonist would be an analog that could select for these endothelial C5a receptors to induce a direct, transient increase in vascular permeability without involving circulating neutrophils. A direct increase in vascular permeability would be useful to augment the delivery of large macromolecules (e.g., monoclonal antibodies) from the blood to surrounding diseased tissue, or across the blood-brain barrier, but not engage neutrophils or their accompanying side effects (adhesion, enzyme release, superoxide release, chemotaxis).
As mentioned, several C-terminal C5a peptide analogs have been produced and studied for the purpose of developing C5a agonists and antagonists. For example, Ember et al. (1992, supra), characterized the biological activities of 22 synthetic C-terminal C5a analogs. The analogs were reported to be full agonists of natural C5a, having in vitro activities characteristic of naturally occurring C5a, including the ability to stimulate ileal contraction (i.e., spasmogenesis) platelet aggregatory activation and neutrophil polarization and chemotaxis. However, the potencies of even the most effective of these analogs was on the order of only 0.01-0.25% that of the natural factor. This level of potency could be obtained with analogs as short as decapeptides, as compared with longer C-terminal peptides that had previously been studied as potential agonists. Morgan et al. (1992, supra) reported that certain of the peptide analogs disclosed by Ember et al. stimulated synthesis of interleukin-6 in human peripheral blood mononuclear cells. Again, however, potency of these peptide analogs was on the order of 0.01-0.1% of either natural or recombinant C5a. Drapeau et al. reported on the pharmacology, metabolism and in vivo cardiovascular and hematologic effects of synthetic C-terminal C5a peptide analogs based on either human or porcine amino acid sequences. These analogs were also found to be agonists of natural C5a, but were disclosed as being at least 1,000-fold less potent than recombinant C5a as measured by competition for C5a binding sites.
Each of the aforementioned reports describes differences among the various peptide analogs with respect to their effectiveness for eliciting specific biological responses associated with C5a. However, the basis for that differential elicitation of biological response was not described with respect to specific structure-function relationships.
C-terminal C5a peptide analogs have also been studied with respect to the ability of such analogs to bind to C5a receptors. Kawai et al. (1992), J. Med. Chem., 35: 220-223, reported on relationships between the hydrophobicity and chirality of residues 70-73 of C-terminal octapeptide analogs and the ability of such analogs to bind to C5a receptors. However, biological responses elicited by these octapeptide analogs was not reported. In other studies, it has been determined that substitution of phenylalanine or tryptophan in positions between 65 and 69 of the human C5a C-terminus could enhance or decrease potency, depending on whether the substitution was made at position 67 or at position 66. In other studies, these observations were corroborated with reports that substitution of phenylalanine for histidine at position 67 substantially increased the potency of a number of C-terminal peptide analogs of human C5a. See Mollison et al. (1991), Agents and Actions, Suppl. 35: 17-21; Or et al. (1992), 35: 402-406; Kohl et al. (1993), Eur. J. Immunol., 23: 646-652. These reports did not address any differences among the various peptide analogs with respect to their effectiveness for eliciting specific biological responses associated with C5a, although it was noted that different tissue or cell types and different species responded differently to the analogs.
Thus, in spite of the numerous C-terminal peptide analogs that have been synthesized and studied to date, no effective peptide-based C5a agonist or antagonist, either general or selective, has been reported. This is in part due to the hitherto unclear relationship between the conformational features of the C-terminal peptides and the biological properties they impart, which has impeded the progress of a rational design strategy for the development of a high-affinity and selective C5a analog.
The general spatial arrangement of the N-terminal region of human C5a (residues 1-63) has been determined on the basis of .sup.1 H-NMR data, but no definable spatial structure could be assigned to the C-terminal "effector" region (residues 64-74), which appears more flexible than the rest of the C5a polypeptide (Zuiderweg et al. (1989), Biochem., 28: 172-185). Flexibility in the C-terminal region appears to play a role in potency and general expression of biological activity because marked changes in potency have been observed when the flexibility in this region was restricted (Ember et al., 1992, supra, reporting that modifications made in the C-terminal portion of the C5a peptide analogs to reduce flexibility of the backbone affected their activity).
To date, however, the structure-function relationship of the C-terminal portion of C5a and its biological activities has not been successfully exploited to develop more potent agonists, nor has it been utilized to produce agonists with selective activities, i.e., the ability to elicit specific biological responses (e.g., spasmogenic response)-in favor of others (e.g., neutrophil-mediated responses). Thus, a need exists to develop more potent C5a agonists and, further, to produce high-affinity C5a analogs having biological response-selective agonistic activity. Such compounds will find broad utility in treating immunocompromised patients, preferably without inflammatory side effects, and as high affinity templates for the development of antagonists to modulate pathological diseases associated with the proinflammatory activities of C5a.